In situ assay for RNA decapping in tumors Abstract This project aims to introduce a new histotechnology to assess tumor cell stress and cell death propensity, which can be useful in evaluating cancer response to therapy. The adaptive response of tumor cells to stress and their survival in hostile environments are signaled by the levels of mRNA decay. Increased decay dumps important mRNAs and marks cells in distress. Conversely, decay blockage is highly protective. It stabilizes active mRNAs in cancer cells, supports metabolic stability and successful adaptation of the tumor. The major RNA destruction pathway is 5?-3? mRNA decay. Its defining step is decapping, which removes the N7-methyl-guanosine cap from 5? RNA ends. RNA decapping is the rate-determining step and the best indicator of the decay activity. Although mRNA decay is a key process in tumor survival, at present it cannot be imaged and assessed in tissue sections. In the absence of specific histotechnologies, it is currently studied by using bulk biochemical approaches which have limited value in heterogeneous pathology samples. In this project we will overcome this obstacle and will develop a new and enabling technology for molecular analysis of tumor cells. We will introduce the first assay for in situ assessment of 5?-3? mRNA decay pathway via specific labeling of its decapping step in tissue sections. The project will create a new molecular histopathology tool and an advantageous product with wide applicability in studies of cancer and drug development where assessments of mRNA decay are essential. The proposal will reach these Specific Aims: 1. To develop the first methodology for selective detection of RNA decapping in tissue sections. The approach will label the key event of this pathway and visualize the characteristic RNA cleavage activity exposing 5? terminal monophosphates. 2. To test and optimize the new labeling approach in tissue sections by using several models with active RNA decay reaction including glioblastoma. To validate the new assay specificity and ensure its adequate sensitivity and reliable robust detection. 1